RESUMO
BACKGROUND: Transcription factor EGR3 (Early Growth Response 3) is a little-studied member of the EGR family that is highly expressed in human prostate tumours compared with normal tissue. Its function in prostate cancer, however, is unknown. METHODS: Stable shRNA silencing was achieved in naturally overexpressing prostate cancer cells, followed by Affymetrix expression analysis. Fold changes of ⩾2 and ⩽-2 were considered valid and t-tests P-values of ⩽0.01 were considered statistically significant. Potential EGR3 target genes were validated by real-time qPCR, chromatin immunoprecipitation, and gain-of-function experiments. Promoter analysis confirmed the presence of consensus binding sites in the promoters of target genes. RESULTS: Early Growth Response 3 regulates the expression of â¼330 genes, 35% of which are involved in immune responses and inflammatory processes, and 15% crosstalk with the NF-κB signalling pathway. In particular, EGR3 induces the expression of over 50 secreted cytokines, growth factors, and matrix remodelling factors. Two interleukins of great relevance to prostate cancer, IL6 and IL8, were further validated as EGR3 target genes: both promoters contain EGR consensus binding sites and are pulled down in intact cells by EGR3 chromatin immunoprecipitation. Silencing of EGR3 decreased IL6 and IL8 expression, whereas overexpression of EGR3 in nontransformed cells induced IL6 and IL8 expression. CONCLUSIONS: Chronic inflammation plays a critical role in prostate cancer and elevated production of pro-inflammatory cytokines IL8 and IL6, in particular, contributes to disease progression and to the onset of castration resistance. It is shown for the first time that EGR3 is involved in the upregulation of both IL6 and IL8. Together with our previous observation that EGR3 is highly expressed in prostate tumours compared with normal tissue and strongly correlates with IL6 and IL8 expression in clinical samples, the present study suggests that EGR3 promotes excessive production of IL6 and IL8 observed during the progression of prostate cancer.
Assuntos
Proteína 3 de Resposta de Crescimento Precoce/fisiologia , Inflamação/genética , Interleucina-6/genética , Interleucina-8/genética , Neoplasias da Próstata/genética , Perfilação da Expressão Gênica , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Análise em Microsséries , Ativação Transcricional , Transfecção , Células Tumorais CultivadasRESUMO
The PTEN tumour suppressor and pro-apoptotic gene is frequently mutated in human cancers. We show that PTEN transcription is upregulated by Egr-1 after irradiation in wild-type, but not egr-1-/-, mice in vivo. We found that Egr-1 specifically binds to the PTEN 5' untranslated region, which contains a functional GCGGCGGCG Egr-1-binding site. Inducing Egr-1 by exposing cells to ultraviolet light upregulates expression of PTEN messenger RNA and protein, and leads to apoptosis. egr-1-/- cells, which cannot upregulate PTEN expression after irradiation, are resistant to ultraviolet-light-induced apoptosis. Therefore, Egr-1 can directly regulate PTEN, triggering the initial step in this apoptotic pathway. Loss of Egr-1 expression, which often occurs in human cancers, could deregulate the PTEN gene and contribute to the radiation resistance of some cancer cells.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces , Monoéster Fosfórico Hidrolases/genética , Monoéster Fosfórico Hidrolases/metabolismo , Transdução de Sinais/efeitos da radiação , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas Supressoras de Tumor/genética , Proteínas Supressoras de Tumor/metabolismo , Animais , Apoptose/fisiologia , Apoptose/efeitos da radiação , Células Cultivadas , Derme/citologia , Proteína 1 de Resposta de Crescimento Precoce , Etoposídeo/farmacologia , Fibroblastos/citologia , Raios gama , Regulação Neoplásica da Expressão Gênica/fisiologia , Regulação Neoplásica da Expressão Gênica/efeitos da radiação , Humanos , Glândulas Mamárias Animais/citologia , Camundongos , Camundongos Knockout , Neoplasias/fisiopatologia , Inibidores da Síntese de Ácido Nucleico/farmacologia , PTEN Fosfo-Hidrolase , Regiões Promotoras Genéticas/genética , RNA Mensageiro/análise , Transdução de Sinais/fisiologia , Raios UltravioletaRESUMO
p73, a transcription factor rarely mutated in cancer, regulates a subset of p53 target genes that cause cells to respond to genotoxic stress by growth arrest and apoptosis. p73 is produced in two main forms; only TAp73 reiterates the roles of p53, while DeltaNp73 can be oncogenic in character. We show that the TAp73 form produced by TP73 P1 promoter has five distinct Egr1-binding sites, each contributing to the transcriptional upregulation of TAp73 by Egr1 in several cell types. In contrast, TP73 P2 promoter transcribes DeltaNp73, is not induced by Egr1, but is induced by TAp73 and p53. Induction of TAp73 by genotoxic stress requires Egr1 in mouse in vivo. Newly discovered non-consensus p53-binding sites in p73, p53 and Egr1 promoters reveal inter-regulating networks and sustained expression by feedback loops in response to stress, resulting in prolonged expression of the p53 family of genes and efficient apoptosis.
Assuntos
Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Regulação Neoplásica da Expressão Gênica , Proteínas Nucleares/metabolismo , Transdução de Sinais , Proteína Supressora de Tumor p53/metabolismo , Proteínas Supressoras de Tumor/metabolismo , Animais , Antineoplásicos Fitogênicos/farmacologia , Apoptose , Linhagem Celular Tumoral , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce/genética , Etoposídeo/farmacologia , Raios gama , Humanos , Camundongos , Modelos Biológicos , Proteínas Nucleares/genética , Regiões Promotoras Genéticas , Fatores de Tempo , Transcrição Gênica , Transfecção , Proteínas Supressoras de Tumor/genética , Regulação para CimaRESUMO
We have previously found that epidermal growth factor (EGF) mediates growth through the Jun N-terminal kinase/stress-activated kinase (JNK/SAPK) pathway in A549 human lung carcinoma cells. As observed here, EGF treatment also greatly enhances the tumorigenicity of A549 cells, suggesting an important role for JNK in cancer cell growth (F. Bost, R. McKay, N. Dean, and D. Mercola, J. Biol. Chem. 272:33422-33429, 1997). Several isoforms families of JNK, JNK1, JNK2, and JNK3, have been isolated; they arise from alternative splicing of three different genes and have distinct substrate binding properties. Here we have used specific phosphorothioate oligonucleotides targeted against the two major isoforms, JNK1 and JNK2, to discriminate their roles in EGF-induced transformation. Multiple antisense sequences have been screened, and two high-affinity and specific candidates have been identified. Antisense JNK1 eliminated steady-state mRNA and JNK1 protein expression with a 50% effective concentration (EC50) of <0.1 microM but did not alter JNK2 mRNA or protein levels. Conversely, antisense JNK2 specifically eliminated JNK2 steady-state mRNA and protein expression with an EC50 of 0.1 microM. Antisense JNK1 and antisense JNK2 inhibited by 40 and 70%, respectively, EGF-induced total JNK activity, whereas sense and scrambled-sequence control oligonucleotides had no effect. The elimination of mRNA, protein, and JNK activities lasted 48 and 72 h following a single Lipofectin treatment with antisense JNK1 and JNK2, respectively, indicating sufficient duration for examining the impact of specific elimination on the phenotype. Direct proliferation assays demonstrated that antisense JNK2 inhibited EGF-induced doubling of growth as well as the combination of active antisense oligonucleotides did. EGF treatment also induced colony formation in soft agar. This effect was completely inhibited by antisense JNK2 and combined-antisense treatment but not altered by antisense JNK1 alone. These results show that EGF doubles the proliferation (growth in soft agar as well as tumorigenicity in athymic mice) of A549 lung carcinoma cells and that the JNK2 isoform but not JNK1 is utilized for mediating the effects of EGF. This study represents the first demonstration of a cellular phenotype regulated by a JNK isoform family, JNK2.
Assuntos
Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Transformação Celular Neoplásica , Proteínas Quinases Ativadas por Mitógeno , Proteínas Quinases/metabolismo , Ágar , Animais , Proteínas Quinases Dependentes de Cálcio-Calmodulina/genética , Divisão Celular , Fator de Crescimento Epidérmico/farmacologia , Feminino , Expressão Gênica , Humanos , Isoenzimas , Proteínas Quinases JNK Ativadas por Mitógeno , Camundongos , Camundongos Nus , Proteína Quinase 9 Ativada por Mitógeno , Oligonucleotídeos , Oligonucleotídeos Antissenso , Proteínas Quinases/genética , RNA Antissenso , RNA Mensageiro , Fatores de Tempo , Células Tumorais Cultivadas , Raios UltravioletaRESUMO
In resting cells, c-Jun is phosphorylated on five sites. Three of these sites reside next to its DNA binding domain and negatively regulate DNA binding. In response to expression of oncogenic Ha-Ras, phosphorylation of these sites decreases, while phosphorylation of two other sites within c-Jun's activation domain is greatly enhanced. Phosphorylation of these residues, serines 63 and 73, stimulates the transactivation function of c-Jun and is required for oncogenic cooperation with Ha-Ras. We now show that the same changes in c-Jun phosphorylation are elicited by a variety of transforming oncoproteins with distinct biochemical activities. These oncoproteins, v-Sis, v-Src, Ha-Ras, and Raf-1, participate in a signal transduction pathway that leads to increased phosphorylation of serines 63 and 73 on c-Jun. While oncogenic Ha-Ras is a constitutive stimulator of c-Jun activity and phosphorylation, the normal c-Ha-Ras protein is a serum-dependent modulator of c-Jun's activity. c-Jun is therefore a downstream target for a phosphorylation cascade involved in cell proliferation and transformation.
Assuntos
Proteínas Oncogênicas Virais/metabolismo , Proteínas Tirosina Quinases/metabolismo , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de Sinais , Células 3T3 , Sequência de Aminoácidos , Animais , Linhagem Celular , Transformação Celular Neoplásica , Eletroforese em Gel de Poliacrilamida , Metionina/metabolismo , Camundongos , Mutação , Proteína Oncogênica p21(ras)/metabolismo , Proteína Oncogênica pp60(v-src)/metabolismo , Proteínas Oncogênicas v-sis , Fosfatos/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas/metabolismo , Proteínas Proto-Oncogênicas c-jun/genética , Proteínas Proto-Oncogênicas c-jun/isolamento & purificação , Proteínas Proto-Oncogênicas c-raf , Proteínas Oncogênicas de Retroviridae/metabolismo , Serina , Ativação TranscricionalRESUMO
6-[3-(1-Adamantyl)-4-hydroxyphenyl]-2-naphthalene carboxylic acid (AHPN or CD437), originally identified as a retinoic acid receptor gamma-selective retinoid, was previously shown to induce growth inhibition and apoptosis in human breast cancer cells. In this study, we investigated the role of AHPN/CD437 and its mechanism of action in human lung cancer cell lines. Our results demonstrated that AHPN/CD437 effectively inhibited lung cancer cell growth by inducing G0/G1 arrest and apoptosis, a process that is accompanied by rapid induction of c-Jun, nur77, and p21(WAF1/CIP1). In addition, we found that expression of p53 and Bcl-2 was differentially regulated by AHPN/CD437 in different lung cancer cell lines and may play a role in regulating AHPN/CD437-induced apoptotic process. On constitutive expression of the c-JunAla(63,73) protein, a dominant-negative inhibitor of c-Jun, in A549 cells, nur77 expression and apoptosis induction by AHPN/CD437 were impaired, whereas p21(WAF1/CIP1) induction and G0/G1 arrest were not affected. Furthermore, overexpression of antisense nur77 RNA in A549 and H460 lung cancer cell lines largely inhibited AHPN/CD437-induced apoptosis. Thus, expression of c-Jun and nur77 plays a critical role in AHPN/CD437-induced apoptosis. Together, our results reveal a novel pathway for retinoid-induced apoptosis and suggest that AHPN/CD437 or analogs may have a better therapeutic efficacy against lung cancer.
Assuntos
Apoptose , Inibidores do Crescimento/farmacologia , Retinoides/farmacologia , Carcinoma Pulmonar de Células não Pequenas , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/biossíntese , Proteínas de Ligação a DNA/biossíntese , Fase G1 , Humanos , Neoplasias Pulmonares , Membro 1 do Grupo A da Subfamília 4 de Receptores Nucleares , Proteínas Proto-Oncogênicas c-jun/biossíntese , Receptores Citoplasmáticos e Nucleares , Receptores de Esteroides , Fase de Repouso do Ciclo Celular , Fatores de Transcrição/biossíntese , Tretinoína/farmacologia , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/biossínteseRESUMO
Recent studies are reviewed indicating that the transcription factor early growth response-1 (Egr1) is a direct regulator of multiple tumor suppressors including TGFbeta1, PTEN, p53, and fibronectin. The downstream pathways of these factors display multiple nodes of interaction with each other, suggesting the existence of a functional network of suppressor factors that serve to maintain normal growth regulation and resist the emergence of transformed variants. Paradoxically, Egr1 is oncogenic in prostate cancer. In the majority of these cancers, PTEN or p53 is inactive. It is suggested that these defects in the suppressor network allow for the unopposed induction of TGFbeta1 and fibronectin, which favor transformation and survival of prostate tumor epithelial cells, and explain the role of Egr1 in prostate cancer. Egr1 is a novel and logical target for intervention by gene therapy methods, and targeting methods are discussed.
Assuntos
Proteína 1 de Resposta de Crescimento Precoce/metabolismo , Fibronectinas/metabolismo , Regulação Neoplásica da Expressão Gênica , Genes p53/genética , Terapia Genética/métodos , PTEN Fosfo-Hidrolase/metabolismo , Neoplasias da Próstata/terapia , Fator de Crescimento Transformador beta/metabolismo , Proteína 1 de Resposta de Crescimento Precoce/genética , Células Epiteliais/metabolismo , Humanos , Masculino , Modelos Genéticos , Inibidor 1 de Ativador de Plasminogênio/metabolismo , Neoplasias da Próstata/metabolismo , Fator de Crescimento Transformador beta1RESUMO
Autocrine stimulation by platelet-derived growth factor-B (PDGF-B)-like factors has been widely implicated as an important mechanism in the cause and/or maintenance of a variety of human tumors. However, normal human cells appear to be resistant to transformation by PDGF-B-like molecules, and a direct demonstration of the tumor-promoting or tumor-maintaining property of a PDGF-B autocrine system is lacking. T98G human glioblastoma cells are nontumorigenic in athymic mice. We show that these cells express predominantly PDGF-beta type receptors and continuously secrete small amount of PDGF-B/c-sis. Addition of suramin or specific anti-PDGF-B/v-sis antibody inhibits proliferation in culture. Conversely, multiple clonal lines that stably overexpress PDGF-B/v-sis (T98Gsis cells) exhibit a striking 200-250% increased proliferation rate and an enhanced colony-forming frequency in soft agar. Clonal lines with stable expression of PDGF-B/v-sis (T98Gsis cells) reliably (80%) develop tumors in 4-6 weeks, whereas the empty-vector control cells are nontumorigenic. Moreover, in some cases, T98Gsis cells disseminate to form bilateral and multifocal pulmonary metastases. The results show that T98G cells contain functional PDGF receptors that, upon sufficient stimulation, can cause greatly increased mitogenic response, which may account for the development of the malignant phenotype. Metastatic tumor formation in athymic mice by PDGF stimulation has not been reported previously. The mechanism may depend on preexisting changes such as the lost p53 function of these cells. T98Gsis cells provide a model of growth factor-dependent tumorigenesis and metastases, which may be helpful in elucidating these relationships.
Assuntos
Glioblastoma/patologia , Fator de Crescimento Derivado de Plaquetas/fisiologia , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Oncogênicas de Retroviridae/fisiologia , Animais , Anticorpos Antineoplásicos/farmacologia , Especificidade de Anticorpos , Divisão Celular/fisiologia , Transformação Celular Neoplásica , Feminino , Glioblastoma/metabolismo , Glioblastoma/secundário , Humanos , Neoplasias Pulmonares/metabolismo , Neoplasias Pulmonares/patologia , Neoplasias Pulmonares/secundário , Masculino , Camundongos , Camundongos Nus , Pessoa de Meia-Idade , Proteínas Oncogênicas v-sis , Fenótipo , Fator de Crescimento Derivado de Plaquetas/biossíntese , Fator de Crescimento Derivado de Plaquetas/genética , Proteínas Proto-Oncogênicas/biossíntese , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas c-sis , Proteínas Oncogênicas de Retroviridae/biossíntese , Proteínas Oncogênicas de Retroviridae/genética , Transfecção , Células Tumorais CultivadasRESUMO
Human HT1080 fibrosarcoma cells, subclone H4, express little or no Egr-1 (Zif/268, Krox 24), an early growth response gene encoding a transcription factor. Phorbol ester (but not serum) treatment only can elicit a small increase in Egr-1 expression in H4, in contrast to the normally rapid, high transient expression of Egr-1 observed after the addition of a wide range of stimulating agents to normal or immortalized cell lines. Because several human tumor cell lines express little Egr-1, we tested the hypothesis that this loss was causal to transformation. We report here that the expression of exogenous mouse Egr-1 in H4 cells inhibits transformed growth in a dose-dependent manner and significantly suppresses tumorigenicity in athymic mice. By overexpression of the fragment in Egr-1 that is responsible for its DNA-binding activity, the zinc-finger domain, we show that this domain has a similar activity. Moreover, the expression of antisense mRNA encoding the DNA-binding domain increases the transformed character of the H4 cells. One possible conclusion is that endogenous Egr-1-like genes perform growth-regulatory functions. Other human tumor lines are also growth suppressed by Egr-1 overexpression including ZR-75-1 breast carcinoma, U251 glioblastoma, and to a lesser extent, SAOS-2 osteosarcoma cells. These results are surprising in light of the "early growth response" character of Egr-1 but extend our earlier report of suppression of growth in v-sis-transformed NIH3T3 cells.
Assuntos
DNA de Neoplasias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Fibrossarcoma/patologia , Proteínas Imediatamente Precoces , Fatores de Transcrição/metabolismo , Animais , Sequência de Bases , Sítios de Ligação , Divisão Celular/fisiologia , Transformação Celular Neoplásica/genética , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Fibrossarcoma/genética , Fibrossarcoma/metabolismo , Humanos , Camundongos , Dados de Sequência Molecular , RNA Antissenso/genética , Fatores de Transcrição/genética , Células Tumorais Cultivadas , Dedos de Zinco/genéticaRESUMO
v-sis is the oncogene of simian sarcoma virus, but whether tumor growth is maintained by v-sis expression alone or requires additional changes is unknown. To distinguish these possibilities we studied a model of reversible transformation including tumorigenicity using NIH3T3 cells bearing a metallothionein promoter-v-sis construction. Cells subcultured from 10 out of 18 tumors from athymic mice, all less than 0.1 g and less than or equal to 21 days in age, reverted to a normal phenotype but exhibited transformation upon addition of zinc as judged by morphology, growth rate, saturation density and anchorage independence of growth. Thus, activation of v-sis alone is sufficient for initiation and early autocrine-based growth of tumors. However, the cells from the remaining and predominantly larger, 0.5 +/- 0.7 g, tumors did not revert and exhibited zinc-independent transformation as judged by the same criteria. Southern analysis and examination of the regulation of v-sis product expression in cells derived from these tumors showed no change in zinc-dependent and reversible regulation of v-sis sequences. These results suggest that subsequent tumor growth strongly favors acquisition of additional irreversible change(s) in the tumor cell genome at high frequency (44%). Thus an early event of a multistep process stimulated by v-sis-dependent transformation best accounts for the sum of results.
Assuntos
Transformação Celular Neoplásica , Oncogenes , Proteínas Oncogênicas de Retroviridae/genética , Células 3T3 , Animais , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Camundongos Endogâmicos BALB C , Transplante de Neoplasias , Proteínas Oncogênicas v-sis , Zinco/farmacologiaRESUMO
The transcription factor Egr-1, stimulates the activity of a number of genes and inhibits other genes, by binding to the sequence GCGGGGGCG in 5' enhancer regions. However, the functions of Egr-1 are obscure in spite of its rather ubiquitous expression. Egr-1 may play a role in proliferation in mitogen-stimulated cells but its expression is also correlated with the differentiated state in teratocarcinoma cells. The constitutive expression of Egr-1 appears to have little effect on the growth rate of normal immortalized cell-lines. We show that in NIH3T3 cells that are conditionally transformed by the expression of v-six, the presence of Egr-1 is inhibitory to the production of transformed colonies (foci) and to growth in soft agar. In addition, the first appearance of tumors in nu/nu mice is delayed in tumorigenicity tests with cells that over-express Egr-1 and tumor growth is suppressed compared to control cells. We used a series of fragments of Egr-1 cloned into expression vectors to show that not only full length, but also truncated Egr-1 fragments inhibit colony formation. Using deletion mutants, we observed that this inhibitory activity is dependent on the presence of the DNA-binding 'zinc-finger' region. Wilm's tumor protein, WT1, (known to be a tumor suppressor gene) that exhibits the same DNA binding activity is also inhibitory. In contrast, colony formation is stimulated by an Egr-1 antisense RNA-expressing plasmid, since colonies grow rapidly and the colony-forming frequency is higher than in the presence of v-sis alone. We conclude that proteins containing the Egr-1 'zinc-finger' domain can bind to the regulatory regions of one or more genes that are required for the transformation of fibroblasts by v-sis thus inhibiting transformation. One function for Egr-1 implied by these results is the restraint of transformed growth in mitogen-stimulated cells.
Assuntos
Transformação Celular Neoplásica , Proteínas de Ligação a DNA/fisiologia , Proteínas Imediatamente Precoces , Proteínas Oncogênicas de Retroviridae/metabolismo , Fatores de Transcrição/fisiologia , Células 3T3/patologia , Animais , Sequência de Bases , Adesão Celular , Divisão Celular , Transformação Celular Neoplásica/efeitos dos fármacos , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Nus , Dados de Sequência Molecular , Mutação , Transplante de Neoplasias , Proteínas Oncogênicas v-sis , RNA Antissenso/metabolismo , RNA Antissenso/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ensaio Tumoral de Célula-Tronco , Dedos de ZincoRESUMO
The expression of fos protein was examined in 30 cases of human osteosarcoma as formalin-fixed and paraffin-embedded tissue sections using two monoclonal antibodies and, for five cases, as frozen sections using 3 polyclonal antibodies. Nuclear antibody labeling intensities were determined either by visual scoring (3 pathologists) or by microdensitometry and included over 700 tumor, 350 normal and 150 benign tissue observations. Visual scores were shown to be linear with optical density with a correlation coefficient of 0.97. Analysis of the osteosarcoma cases at one antibody concentration revealed two groups: a minority (39%) with a low average visual score of 2 +/- 0.2 which was very similar to benign and normal tissues, and a majority (61%) with an average score of 3.1 +/- 0.3. The difference is highly significant (t-test), P less than or equal to 0.01. The results were supported by an analysis of partial immunotitration curves using a curve-fitting procedure which yielded estimates of FOSo (maximum relative fos protein concentration) which were ca. 150% increased for the majority tumor group compared to the minority group or benign or normal tissues. In previous studies of v-sis transformed cells, which exhibit ca. 300% over-expression of c-fos protein as observed here, antisense techniques were used to show that over-expression leads to increased growth and loss of contact inhibition. Thus the combined results suggest that steady state c-fos protein is significantly elevated in, and may contribute to, the aggressive growth properties of a majority of human osteosarcomas.
Assuntos
Osteossarcoma/metabolismo , Proteínas Proto-Oncogênicas/metabolismo , Proto-Oncogenes , Anticorpos Monoclonais , Divisão Celular , Relação Dose-Resposta Imunológica , Expressão Gênica , Humanos , Imuno-Histoquímica , Proteínas Nucleares/metabolismo , Osteossarcoma/patologia , Proto-Oncogene Mas , Proteínas Proto-Oncogênicas c-fos , Células Tumorais CultivadasRESUMO
The human fibrosarcoma cell line, HT1080, clone H4, was used to determine if the transformation suppressive functions of p53 and Egr-1 have the same underlying mechanism. This cell line expresses only mutant p53 and no detectable Egr-1. H4 clones stably expressing Egr-1 are less transformed in proportion to the level of Egr-1 expressed, acting through the induction of the TGFbeta1 gene. Here, H4 cells and the highest Egr-1 expressing clone were transfected with a vector expressing normal human p53 to derive stable clones expressing p53. The expression of p53 in H4 cells inhibited transformed growth and reduced tumorigenicity. The effect of coexpression of both p53 and Egr-1 was additive, producing cell lines with 30% of normal growth rate and sevenfold reduced tumorigenicity compared with control lines. These results indicated that each factor may act independently by different pathways, although each additively increased the level of p21WAF1 cell cycle inhibitor. However, exposure of the H4-derived cells to UV-C irradiation produced contrasting effects. Cell cycle analyses showed that the presence of p53 was associated with loss of the G1 and S cells to apoptosis after irradiation. In contrast, the expression of Egr-1 increased entry into S/G2 phase of the cell cycle with little apoptosis via a mechanism involving elevated FAK and low caspase activities. Apoptosis was observed only in the cell lines that expressed no Egr-1, especially those expressing wt-p53, and was preceded by high caspase activity. In summary, Egr-1 suppressed transformation and counteracted apoptosis by the coordinated activation of TGFbeta1, FN, p21 and FAK, leading to enhanced cell attachment and reduced caspase activity. In the doubly expressing cell line, the survival effect of Egr-1 was dominant over the apoptotic effect of p53.
Assuntos
Apoptose , Transformação Celular Neoplásica , Proteínas de Ligação a DNA/metabolismo , Proteínas Imediatamente Precoces , Proteínas Quinases JNK Ativadas por Mitógeno , Quinases de Proteína Quinase Ativadas por Mitógeno , Proteínas Quinases Ativadas por Mitógeno , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p53/antagonistas & inibidores , Proteína Supressora de Tumor p53/metabolismo , Apoptose/efeitos da radiação , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Testes de Carcinogenicidade , Caspase 3 , Caspases/metabolismo , Adesão Celular , Moléculas de Adesão Celular/metabolismo , Ciclo Celular/efeitos da radiação , Divisão Celular/efeitos da radiação , Inibidor de Quinase Dependente de Ciclina p21 , Ciclinas/metabolismo , Fragmentação do DNA/efeitos da radiação , Proteínas de Ligação a DNA/genética , Proteína 1 de Resposta de Crescimento Precoce , Fibrossarcoma , Quinase 1 de Adesão Focal , Proteína-Tirosina Quinases de Adesão Focal , Humanos , MAP Quinase Quinase 4 , Proteína Quinase 3 Ativada por Mitógeno , Mutação , Proteínas Quinases/metabolismo , Proteínas Tirosina Quinases/metabolismo , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Transdução de Sinais , Fatores de Transcrição/genética , Transfecção , Células Tumorais Cultivadas , Proteína Supressora de Tumor p53/genética , Raios UltravioletaRESUMO
In this paper we have investigated the role of Egr-1 in B cell growth regulation by examining the gene expression in a panel of B cell lines, including both EBV genome negative and EBV carrying cell lines. Egr-1 expression correlates with the cellular phenotype and the specific pattern of viral latency established within the individual cell lines. Thus, constitutive activation of Egr-1 gene is invariably associated with unrestricted expression of viral latent genes in all group III EBV genome carrying cell lines. In contrast, Egr-1 expression is abrogated in group I Burkitt tumor cells, irrespective of the EBV genome carrying status. Activated viral gene expression associated with phenotypic conversion of group I cell lines in to group II or III restores the Egr-1 gene expression. Several forms of EGR-1 protein are found within the different groups of cell lines, and the binding activity to DNA consensus sequences was investigated. Finally, time course analysis of Egr-1 expression during the early steps of EBV infection in vitro demonstrated that Egr-1 is upregulated within minutes from the initial interaction with the B lymphocyte.
Assuntos
Linfócitos B , Proteínas de Ligação a DNA/genética , Regulação Viral da Expressão Gênica , Herpesvirus Humano 4/genética , Proteínas Imediatamente Precoces , Fatores de Transcrição/genética , Latência Viral , Linfócitos B/citologia , Linfócitos B/virologia , Divisão Celular , Linhagem Celular Transformada , Proteínas de Ligação a DNA/metabolismo , Proteína 1 de Resposta de Crescimento Precoce , Infecções por Herpesviridae/metabolismo , Herpesvirus Humano 4/fisiologia , Humanos , Ativação Linfocitária , Linfoma de Células B/genética , Linfoma de Células B/metabolismo , Fenótipo , RNA Mensageiro/metabolismo , Fatores de Transcrição/metabolismo , Regulação para CimaRESUMO
1. The near ultraviolet circular dichroism of tropomyosin is due to tyrosine and to disulphide bonds. The optical activity of these chromophores can be distinguished by oxidising and reducing the protein. The circular dichroism due to tyrosine is exceptionally intense and is anomalous in that the shape of the spectrum and the wavelength of the maximum are different from those of the absorption spectrum. 2. The intense tyrosyl circular dichroism and the mismatch between circular dichroism and absorption spectra are likely to be due to tyrosine-tyrosine interactions at distances of less than 8 A. 3. The results are analysed in terms of the coiled coil model for tropomyosin and lead to the conclusion that the tyrosines of one helical subunit interact with those of the other. The in-register alignment of the helical chains, with five tyrosines of one chain opposite those of the other, accounts for the tyrosine-tyrosine interactions. 4. The disulphide circular dichroism of oxidized tropomyosin is intense and is consistent with intra-molecular disulphide bonds between helical subunits which can form in the non-staggered model for tropomyosin.
Assuntos
Tropomiosina , Tirosina/análise , Animais , Dicroísmo Circular , Músculos , Conformação Proteica , CoelhosRESUMO
Previous proton magnetic resonance studies of the effects of Ca(II) on the structure of rabbit skeletal muscle troponin--C have shown that Ca(II) binding to the two high affinity sites of troponin--C both directs and stabilizes the folding of much of the structure. Ca(II) binding by the two low affinity sites of troponin--C causes changes in the environment of largely hydrophobic residues. We have now examined the structural changes caused by Mg(II) and by Ca(II) in the presence of excess Mg(II). Successive addition of Mg(II) to metal-free troponin--C leads to broadly similar, but not identical, structural changes to those previously assigned to Ca(II) binding at the high affinity sites. None of the changes previously assigned to Ca(II) binding to the low affinity sites was observed. Since Mg(II) does not bind to the low affinity sites, these results confirm the previous assignments. The spectral differences between Mg(II) and Ca(II) show that the degree of backbone folding and interactions between a group of hydrophobic residues (one or more Val, Leu, Ile; two or more Phe) are different for the two cations. In the presence of excess Mg(II), at a molar ratio that may exist in vivo (approx. 40 : 1 mol ratio Mg(II): Ca(II), titration with Ca(II) leads to a displacement of Mg(II) and to all the structural changes previously observed for Ca(II) alone. However, in the presence of Mg(II) the distinction between high and low affinity sites is blurred as judged by the overlap of the spectral changes associated with each of the binding sites. This result, together with the observation that Mg(II) promotes structural changes different from Ca(II), suggests a structural basis for the observation that the Ca(II) threshold for the activation of tension in some myofibrils is increased in the presence of high Mg(II) concentrations.
Assuntos
Proteínas Musculares , Troponina , Animais , Apoproteínas , Sítios de Ligação , Cálcio/farmacologia , Magnésio/farmacologia , Espectroscopia de Ressonância Magnética , Conformação Proteica/efeitos dos fármacos , Coelhos , Relação Estrutura-AtividadeRESUMO
A relationship was proved between constitutive activity of leukemic cell c-jun-N-terminal kinase (JNK) and treatment failure in AML. Specifically, early treatment failure was predicted by the presence of constitutive JNK activity. The mechanistic origins of this association was sought. A multidrug resistant leukemic cell line, HL-60/ADR, characterized by hyperexpression of c-jun and JNK activity, was transfected with a mutant c-jun vector, whose substrate N-terminal c-jun serines were mutated. Down-regulated expression occurred of c-jun/AP-1-dependent genes, catalase and glutathione-S-transferase (GST) pi, which participate in cellular homeostasis to oxidative stress and xenobiotic exposure. MRP-efflux was abrogated in HL-60/ADR cells with dominant-negative c-jun, perhaps because MRP1 protein expression was also lost. Heightened sensitivity to daunorubicin resulted in cells subjected to this change. Biochemical analysis in 67 primary adult AML samples established a statistical correlation between cellular expression of c-jun and JNK activity, JNK activity with hyperleukocytosis at presentation of disease, and with exuberant MRP efflux. These findings reflect the survival role for c-jun/AP-1 and its regulatory kinase previously demonstrated for yeast in homeostatic response to oxidative stress and in operation of ATP-binding cassette efflux pumps, and may support evolutionary conservation of such function. Thus, JNK and c-jun may be salient drug targets in multidrug resistant AML.
Assuntos
Resistencia a Medicamentos Antineoplásicos/fisiologia , Leucemia Mieloide/enzimologia , Proteínas Quinases Ativadas por Mitógeno/fisiologia , Membro 1 da Subfamília B de Cassetes de Ligação de ATP/metabolismo , Doença Aguda , Células da Medula Óssea/patologia , Divisão Celular , Daunorrubicina , Resistência a Múltiplos Medicamentos/fisiologia , Células HL-60 , Humanos , Proteínas Quinases JNK Ativadas por Mitógeno , Leucemia Mieloide/tratamento farmacológico , Leucemia Mieloide/patologia , Proteínas Quinases Ativadas por Mitógeno/metabolismo , Fosforilação , Proteínas Proto-Oncogênicas c-jun/metabolismo , Transdução de SinaisRESUMO
PURPOSE: EGR-1 is an immediate early gene with diverse functions that include the suppression of growth. EGR-1 is down-regulated many cancer cell types, suggesting a tumor suppressor role, and may critically involve the p53 pathway. The aim of this work was to measure the expression of EGR-1 and the p16/INK4a/ARF-Mdm2-p53 pathway status in fresh human gliomas. EXPERIMENTAL DESIGN: Thirty-one human gliomas with different grades of malignancy were investigated for Egr-1 mRNA and the protein expression, frequency, and spectrum of p53 gene mutations, mdm2 gene amplification, and p16/INK4a/ARF allele loss. RESULTS: The amplification of Mdm2 and the deletion of the p16/INK4a gene was found in 3 and 5 cases, respectively, whereas mutations of p53, including two novel mutations, were observed in 10 other cases. The three types of changes occurred strictly mutually exclusively, emphasizing that these genes operate in a common pathway critical to glioma progression. EGR-1 mRNA was significantly down-regulated in astrocytomas (14.7 +/- 5.1%) and in glioblastomas (33.6 +/- 10.0%) versus normal brain. Overall, EGR-1 mRNA was strongly suppressed (average, 15.2 +/- 13.9%) in 27 of 31 cases (87%), independent of changes in p16/INK4a/ARF and Mdm2; whereas 4 of 31 cases with residual EGR-1 expression as well as the highest EGR-1 variance segregated with p53 mutations. Immunohistochemical analyses confirmed the suppression of EGR-1 protein. CONCLUSIONS: These results indicate that EGR-1 is commonly suppressed in gliomas independent of p16/INK4a/ARF and Mdm2 and that suppression is less crucial in tumors bearing p53 mutations, and these results implicate an EGR-1 growth regulatory mechanism as a target of inactivation during tumor progression.
Assuntos
Neoplasias Encefálicas/genética , Proteínas de Ligação a DNA/genética , Glioma/genética , Proteínas Imediatamente Precoces , Proteínas Nucleares , Proteínas/fisiologia , Fatores de Transcrição/genética , Northern Blotting , Neoplasias Encefálicas/patologia , Inibidor p16 de Quinase Dependente de Ciclina/genética , Análise Mutacional de DNA , DNA de Neoplasias/química , DNA de Neoplasias/genética , Proteínas de Ligação a DNA/metabolismo , Regulação para Baixo , Proteína 1 de Resposta de Crescimento Precoce , Deleção de Genes , Regulação Neoplásica da Expressão Gênica , Glioma/patologia , Humanos , Imuno-Histoquímica , Mutação de Sentido Incorreto , Proteínas/genética , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/fisiologia , Proteínas Proto-Oncogênicas c-mdm2 , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , RNA Neoplásico/genética , RNA Neoplásico/metabolismo , Fatores de Transcrição/metabolismo , Proteína Supressora de Tumor p14ARF , Proteína Supressora de Tumor p53/genética , Proteína Supressora de Tumor p53/fisiologiaRESUMO
The purpose of this study was to determine the safety, toxicity, and antitumor immune response following S.C. immunizations with a mixture of irradiated, autologous tumor cells and autologous fibroblasts that were genetically modified to express the gene for interleukin 2 (IL-2) in patients with colorectal carcinoma. Ten patients were treated with a fixed dose of tumor cells (10(7)) and escalating doses of fibroblasts secreting IL-2 (per 24 h): 100 units (three patients), 200 units (three patients), 400 units (three patients), and 800 units (one patient). Pre- and posttreatment peripheral blood mononuclear cells were evaluated for evidence of antitumor immune responses. Fatigue and/or flu-like symptoms were experienced by seven patients and delayed-type hypersensitivity-like skin reactions were observed at the sites of the second or subsequent vaccinations in five patients. Low frequencies of tumor cytotoxic T-cell precursors (range, 1/190,000-1/1,320,000 peripheral blood mononuclear cells) were detected prior to therapy in four of seven patients. There was a 5-fold increase following treatment in the frequency of tumor cytotoxic T-cell precursors in two of six evaluable patients. Some patients with colorectal cancer have low frequencies of tumor cytotoxic T-cell precursors that may be increased by this well-tolerated form of IL-2 gene therapy, which warrants continued clinical evaluation.
Assuntos
Vacinas Anticâncer/uso terapêutico , Neoplasias Colorretais/terapia , Fibroblastos/metabolismo , Terapia Genética/métodos , Imunoterapia Adotiva/métodos , Interleucina-2/biossíntese , Interleucina-2/genética , Vacinas Anticâncer/imunologia , Transplante de Células , Neoplasias Colorretais/imunologia , Neoplasias Colorretais/patologia , Terapia Combinada , Fibroblastos/fisiologia , Fibroblastos/transplante , Engenharia Genética , Terapia Genética/efeitos adversos , Humanos , Hipersensibilidade Tardia/etiologia , Hipersensibilidade Tardia/imunologia , Leucócitos Mononucleares/imunologia , Leucócitos Mononucleares/efeitos da radiação , Linfócitos T Citotóxicos/imunologia , Linfócitos T Citotóxicos/efeitos da radiação , Linfócitos T Citotóxicos/transplanteRESUMO
Simian sarcoma virus (SSV)-infected NIH-3T3 cells (SSV-NIH-3T3), express a homologue of platelet-derived growth factor, (PDGF) a powerful inducer of the c-fos gene. We have used these cells to test the hypothesis that autocrine stimulation by PDGF-like molecules leads to c-fos expression which is functional in the transformed phenotype. We have transfected SSV-NIH-3T3 cells with a c-fos antisense-RNA expression vector, pSVsof, or control plasmids. pSVsof-transfected cells exhibit markedly decreased c-fos mRNA and protein levels, restored density-dependent growth arrest and reduced (three of five clones) tumorigenicity compared to control lines. The results confirm that c-fos cooperates in the transformed phenotype of SSV-NIH-3T3 cells.